Prophage induction in bacterial strains

1. Research Background

In sequenced bacterial genomes, more than half carry at least one prophage, and some strains carry several prophage regions, collectively occupying 10%–20% of the host genome's sequence space. Prophages are not only one of the main drivers of bacterial genome evolution, but also confer key phenotypes to the host through lysogenic conversion—multiple important virulence factors, such as diphtheria toxin, Shiga toxin, and cholera toxin, are encoded by propages.

The activation (induction) of prophages has a dual significance. On the one hand, it poses a risk: in industrial fermentation and probiotic production, spontaneous or stress-induced prophage induction can cause cell lysis, fermentation collapse, and yield loss. In the safety evaluation of food, feed, and probiotic strains, regulatory agencies focus on whether prophages can be induced, whether they can release infectious particles, and whether they co-carry virulence or resistance genes, which directly relates to the risk of lateral gene transfer. On the other hand, it is a resource: for hosts such as anaerobic bacteria, gut symbiotic bacteria, and actinomycetes, where it is difficult to isolate phages from the environment, inducing their own prophages is often the most efficient way to obtain new phages.

Based on the above needs, Shutong Biotechnology has further launched a lysogenic phage induction service, building upon its existing "Precise Prediction Solution for Bacterial Prephages". We combine bioinformatics prediction with wet experimental induction verification to provide customers with a one-stop solution, from prephage localization, induction condition optimization, excision and particle verification, to infectivity evaluation and genome characterization.

2. Biological Background and Technical Challenges

(1) Solvent source-pyrolysis decision switchAfter temperate bacteriophages enter a lysogenic state, they are silenced by repressor proteins (such as CI in λ phage). When the host experiences DNA damage, RecA is activated by single-stranded DNA to form RecA*, which mediates the autocleavage of LexA with the phage repressor protein, releasing the inhibition and initiating the lysis cycle. This SOS-dependent mechanism is the theoretical basis for most chemical/physical induction protocols.

(2) The presence of cryptic prophagesMany prophages have degenerated into cryptic/defective prophages due to gene loss or inversion during long-term evolution. Although they can still be identified by prediction software on the genome, they cannot produce complete infectious particles.Induction experiments are the sole gold standard for distinguishing between "active prophages" and "cryptic prophages".This is also the fundamental reason why pure bioinformatics prediction cannot replace wet experiments.

(3) Diversity of inducing factorsDifferent bacterial genera and different prophages exhibit highly varying responses to inducing factors. Commonly used methods include mitomycin C (MMC), quinolone antibiotics (norfloxacin, ciprofloxacin), ultraviolet irradiation, oxidative stress such as hydrogen peroxide, heat and cold shock, acid and bile salt stress, nutrient restriction, and certain phage-specific group communication systems (such as arbitrium peptide signaling). Failure to induce a single agent does not necessarily mean that the prophage is uninducible.

(4) Narrow dose windowIf the concentration of the inducer is too low, it will not be enough to trigger the SOS response; if it is too high, it will directly kill the host bacteria and prevent the lysis cycle from being completed. The optimal concentration is often strain-specific and must be optimized strain by strain through a gradient matrix.

(5) The problem of missing indicator bacteriaInduced phage release often fails to infect the original host (lysogenic bacteria possess superinfectious immunity), thus frequently resulting in "visible lysis but no plaques." It is necessary to screen closely related non-lysogenic strains or deimmunized mutants as indicator bacteria, or to employ a detection strategy independent of indicator bacteria.

[Illustration] Figure 1. Schematic diagram of solvent source-fracture decision switch and SOS induction mechanism.

3. Mainstream Induction and Verification Strategies

3.1 Bioinformatics-based prediction first:

(1) Based on tools such as PHASTEST, geNomad, VIBRANT, and CheckV, the whole genome of the strain was scanned to determine the prephage region boundary, attL/attR site, integrase and repressor protein genes.

(2) Based on the genome integrity score, the "intact/suspicious/cryptic" types are initially determined, and the presence of virulence factors and drug resistance genes in the region is simultaneously investigated to provide a target basis for subsequent induction programs and risk assessments.

3.2 Multi-factor induced matrix (mainstream approach):

Using the mitomycin C concentration gradient as the main line, multiple induction conditions such as quinolones, ultraviolet light, and hydrogen peroxide were set in parallel. Combined with different growth stages (early/mid logarithmic) and treatment duration, the induction response was interpreted by OD₆₀₀ lysis curve and culture clarity to lock in the optimal induction window.

3.3 Verification of cleavage at the molecular level:

(1) PCR of attB/attP circular intermediate: Detection of the circular intermediate formed after the excision of the prephage is the most direct molecular evidence of the excision event.

(2) Quantitative analysis of attL/attR deletion PCR and qPCR: Using the host housekeeping gene as an internal reference, the relative copy number enrichment fold of the phage genome after induction was quantified to achieve quantitative evaluation of induction efficiency.

3.4 Validation at the particle level:

After centrifugation, filtration, and PEG concentration, the supernatant from the induced culture was observed by transmission electron microscopy (TEM) with negative staining to confirm whether complete phage particles were formed and to record morphological parameters. After removing free nucleic acids by DNase/RNase treatment, DNA was extracted from the particles and sequenced to confirm the identity of the packaging genome and its terminal structure, while assessing whether there is a risk of universal transduction of host DNA.

3.5 Infectivity verification:

By screening a library of closely related non-lysogenic strains or constructing deimmunized (repressor-deleted) recipient bacteria, the actual infectivity and host spectrum of the induced bacteriophages were confirmed using the double-layer plate method, drop method, and EOP.

4. Core Application Areas

（1）robustness evaluation of industrial and fermentation strainsThe aim is to quantify the frequency of spontaneous induction and the sensitivity to stress induction, assess the risk of fermentation failure and yield decline due to prophage activation in large-scale fermentation, and provide direct evidence for the construction of "prophage-free chassis bacteria".

（2）Safety evaluation of probiotics, food-grade and feed-grade strainsThe purpose is to assess the inducibility and infectivity of the prophage carried by the strain, and whether it may mediate the lateral transfer of virulence/resistance genes, and output experimental data that can be directly used for product application and safety verification.

（3）Safety assessment of genetically modified microorganisms (GMM)The purpose is to detect the activation status of prophages in gene-edited strains and assess the potential risk of exogenous genes spreading to the environmental microbiome along with phage particles.

（4）New phage resource miningTemperate bacteriophages are obtained from hosts that are difficult to isolate from the environment, such as anaerobic bacteria, intestinal symbiotic bacteria, and actinomycetes, and used as starting materials for subsequent engineering modifications (knocking out integrase and repressor to achieve "cleavage").

（5）Basic researchResearch on lysogen switching mechanisms, host-phage co-evolution, bacterial population phenotypic heterogeneity, and phage-mediated gene flow.

（6）Phage tool development: Extracting site-specific recombination systems (integrase and attP/attB sites), transduction vector backbones, endolysins, and depolymerases from prophages.

5. Project Process and Validation

We provide end-to-end services from strain reception to data delivery, ensuring that conclusions are supported by a strong chain of evidence:

（1）Strains receiving and genome analysisComplete strain activation, purity confirmation, and whole genome sequencing; run the prephage prediction workflow and output regional annotation and risk gene screening results.

（2）Induced condition matrix screeningBased on the characteristics of the strain, a multi-factor, multi-concentration, and multi-duration induction matrix was designed to lock in the optimal conditions using growth curves and lysis phenotypes.

（3）Molecular verification: attB/attP circularization PCR and qPCR quantification to confirm the excision event and quantify the induction efficiency.

（4）Particle purification and characterizationAfter concentration and purification of the supernatant, TEM observation and phage genome sequencing were performed to confirm the authenticity and identity of the particles.

（5）Indicator bacteria screening and infectivity assessmentScreening for sensitive indicator bacteria and determining plaque formation ability, host spectrum and EOP.

（6）Report delivery and preservationProvide complete data reports and offer preservation services for induced phages.

[Illustration] Figure 2: Project Flowchart

6. Introduction to Lysogenous Phage Induction Service

6.1 Core services include:

（1）Prephage bioinformatics prediction and annotationRegional location, integrity scoring, integration site identification, and screening for co-carrying virulence/resistance genes (can be ordered separately as an independent service).

（2）Screening and optimization of multi-factor induction conditionsThe standard induction matrix covers multiple pathways of chemical, physical, and nutritional stress.

（3）Quantitative induction efficiencyEvaluation using a dual approach: qPCR relative quantification and plaque counting.

（4）Phage particle purification, electron microscopy and whole genome sequencing: To confirm particle morphology, packaging genome identity, and transduction risks.

（5）Indicator bacteria screening and host spectrum determinationEstablish a stable detection system that can be used for all subsequent experiments.

（6）Prephage knockout and chassis purificationConnect to our bacterial gene editing platform to build robust industrial chassis bacteria for our clients without precursor phages.

（7）Mild bacteriophage "lysis" modification: Connecting to the phage gene editing platform, by knocking out integrase/repressor, the induced mild phage is modified into an obligate lytic type for therapeutic and preventative development.

6.2 Technical Advantages:

（1）Prediction and Experiment: A Dual-Engine ApproachFirst locate the target, then induce it; avoid blind trial and error, significantly shorten the cycle and increase the positive rate.

（2）Multi-factor parallel matrixIt does not rely on a single inducer and still has multiple alternative pathways for difficult strains that are "unresponsive to MMC".

（3）Solutions for scenarios without indicator bacteriaEven when no sensitive indicator bacteria can be found, a triple evidence chain can still be constructed using qPCR quantification, TEM morphology, and particulate genome sequencing to give a clear conclusion of "inducibility".

（4）Integrate with the security assessment systemIt directly connects to our platform for safety assessment of genetically modified microorganisms, safety assessment of natural strains, and identification and typing of microorganisms, and outputs a complete data package that can be used for product application.

（5）Upstream and downstream platform collaborationServices such as in-stock bacterial strains, whole-genome sequencing of bacterial strains, bacterial gene editing, and bacteriophage gene editing can be delivered in combination to form a closed loop of "prediction-induction-characterization-modification".

7. Case Introduction

Case Study: Single-cell level analysis of spontaneous induction by Corynebacterium glutamicum CGP3 prophage

Project Content: To establish an analytical system combining fluorescence reporter assay and single-cell imaging, reveal the spontaneous induction pattern and SOS-dependent mechanism of large prophage CGP3 in the industrial strain Corynebacterium glutamicum, and provide a theoretical basis for constructing prophage-free industrial chassis bacteria.

(1) Research Background

Corynebacterium glutamicum (Corynebacterium glutamicumThis strain is a core production strain in the amino acid industry. Its type strain, ATCC 13032, carries multiple prophage regions in its genome, with CGP3 being the largest at approximately 187 kb. Spontaneous induction of prophages leads to the cessation of growth and lysis of some cells, resulting in latent biomass and yield losses. However, such events occur at extremely low rates in populations, making them difficult to capture using traditional population averaging methods, and their frequency and regulatory mechanisms have remained unclear for a long time.

(2) Scheme design

The research team constructed a reporter strain with CGP3 prophage promoter driving fluorescent protein expression, enabling visualization of CGP3 activation at the single-cell level. They combined flow cytometry to quantify the induced subsets in the population and used microfluidic chips for live-cell time-lapse imaging to track the complete fate of individual cells from induction to lysis. Simultaneously, they used DNA damaging agents such as mitomycin C as positive induction controls and constructed recA deletion mutants to elucidate the role of the SOS pathway.

(3) Experimental conclusions

Under normal culture conditions without external stress, only a very small subset (far less than 1%) of cells exhibited spontaneous CGP3 induction; single-cell imaging showed that these cells subsequently stopped dividing and eventually lysed, indicating that the induction event was lethal to the individual. Application of DNA-damaging agents significantly increased the induction rate, while recA deletion almost completely eliminated spontaneous induction, clearly confirming that the process was mediated by an SOS response. Simultaneous cleavage and circularization of the CGP3 region were detected at the molecular level.

(4) Application significance

This study reveals that "low-proportion but persistent" spontaneous induction is a significant source of latent performance loss in industrial strains, and directly drives the construction of prophage-free chassis strains (such as the C. glutamicum MB001 platform strain, which exhibits superior genetic stability and transformation efficiency after removing all prophages). Its methodology—combining bioinformatics localization, reporter system construction, induction matrix, single-cell quantification, and molecular validation—can be directly transferred to the robustness and safety evaluation of probiotics, fermentation strains, and genetically modified strains, forming the core technology of our lysogenic phage induction service.

8. References

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[2] Ptashne M. (2004). A Genetic Switch: Phage Lambda Revisited (3rd ed.). Cold Spring Harbor Laboratory Press.

[3] Fortier LC, Sekulovic O. (2013). Importance of prophages to evolution and virulence of bacterial pathogens. Virulence, 4(5), 354-365.

[4] Howard-Varona C, Hargreaves KR, Abedon ST, Sullivan MB. (2017). Lysogeny in nature: mechanisms, impact and ecology of temperate phages. The ISME Journal, 11(7), 1511-1520.

[5] Nanda AM, Heyer A, Krämer C, Grünberger A, Kohlheyer D, Frunzke J. (2014). Analysis of SOS-induced spontaneous prophage induction in Corynebacterium glutamicum at the single-cell level. Journal of Bacteriology, 196(1), 180-188.

[6] Nanda AM, Thormann K, Frunzke J. (2015). Impact of spontaneous prophage induction on the fitness of bacterial populations and host-microbe interactions. Journal of Bacteriology, 197(3), 410-419.

[7] Baumgart M, et al. (2013). Construction of a prophage-free variant of Corynebacterium glutamicum ATCC 13032 for use as a platform strain for basic research and industrial biotechnology. Applied and Environmental Microbiology, 79(19), 6006-6015.

[8] Zhang X, McDaniel AD, Wolf LE, Keusch GT, Waldor MK, Acheson DW. (2000). Quinolone antibiotics induce Shiga toxin-encoding bacteriophages, toxin production, and death in mice. The Journal of Infectious Diseases, 181(2), 664-670.

[9] Erez Z, et al. (2017). Communication between viruses guides lysis-lysogeny decisions. Nature, 541(7638), 488-493.

[10] Wishart DS, et al. (2023). PHASTEST: faster than PHASTER, better than PHAST. Nucleic Acids Research, 51(W1), W443-W450.

[11] Kieft K, Zhou Z, Anantharaman K. (2020). VIBRANT: automated recovery, annotation and curation of microbial viruses, and evaluation of viral community function from genomic sequences. Microbiome, 8, 90.

[12] Nayfach S, et al. (2021). CheckV assesses the quality and completeness of metagenome-assembled viral genomes. Nature Biotechnology, 39, 578-585.

[13] Camargo AP, et al. (2024). Identification of mobile genetic elements with geNomad. Nature Biotechnology, 42, 1303-1312.

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